Mutagenic oxidative DNA base damage increases with age in prostatic tissue. Various factors may influence this increase including: increased production of reactive oxygen species, increased susceptibility to oxidative stress, alterations in detoxifying enzyme levels or defects in DNA repair. Using LC/MS and GC/MS, we show increased levels of oxidative DNA base lesions, 8-hydroxyguanine (8-oxoG), 8-oxoadenine (8-oxoA) and 5-hydroxycytosine (5OHC) over the baseline in PC-3 and DU-145 prostate cancer cells following exposure to ionizing radiation and a repair period. Nuclear extracts from PC-3 and DU-145 prostate cancer cell lines are defective in the incision of 8-oxoG, 5OHC and thymine glycol (TG) relative to the non-malignant prostate cell line. Consistent with reduced expression of OGG1 2a, incision of 8-oxoG is reduced in PC-3 and DU-145 mitochondrial extracts. We also show a correlation between severely defective incision of TG and 5OHC and reduced levels of NTH1 in PC-3 mitochondria. The antioxidant enzymes, glutathione peroxidase (GPx), catalase, and superoxide dismutases (SOD1, SOD2), have altered expression patterns in these cancer cell lines. Genetic analysis of the OGG1 gene reveals that both PC-3 and DU-145 cell lines harbor polymorphisms associated with a higher susceptibility to certain cancers. These data suggest that the malignant phenotype in PC-3 and DU-145 cell lines may be associated with defects in base excision repair (BER) and alterations in expression of antioxidant enzymes. Prostate cancer, is the most prevalent form of cancer among American men and is the second leading cause of their cancer mortality. In the United States, prostate cancer is one of the fastest growing cancers in terms of incidence among American men. While certain factors including: dietary, genetic, lifestyle and environmental are associated with prostate cancer risk, the molecular mechanisms underlying the etiology of the disease are largely unknown. Several genes associated with heritable forms of prostate cancer have been identified and somatic alterations in these genes are presumed to set the stage for the development and/or progression of the disease. To this end, it has been shown that hypermethylation of the -class glutathione S-transferase gene (GSTP1) promoter region inhibits transcription of the gene and is associated with prostate cancer development [4-6]. The GSTP1 gene product probably protects genomic DNA in prostate cells from the deleterious effects of genotoxic agents. Environmental carcinogens such as polycyclic aromatic hydrocarbons may play a role in the etiology of prostate cancer since 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine has been shown to induce prostate cancer in rats. Reactive oxygen species (ROS), most notably the hydroxyl radicals, generated endogenously by cellular metabolism are known to cause oxidative DNA damage that has been implicated in prostate carcinogenesis. Research on the development of prostate cancer suggests that symptomatic and asymptomatic chronic and acute inflammation occurs in the prostate over the life span and acts in synergy with other factors to cause injury to prostatic epithelium. In response to this injury, cellular proliferation occurs followed by oxidative stress related to the ongoing inflammatory process that may in turn result in high rates of oxidative damage to DNA. Furthermore, Bostwick et al. has reported low levels of SOD1, SOD2, and catalase in prostate intraepithelial neoplasia and prostate cancer relative to benign prostate epithelium thereby implicating oxidative DNA damage in prostate carcinogenesis. There is also a significant increase in the proportion of mutagenic oxidatively induced DNA base lesions, 8-hydroxyadenine (8-oxoA), and 8-hydroxyguanine (8-oxoG) in malignant prostatic tissue as well as an increase in the levels of these lesions in benign prostatic tissue with aging. The existence of OGG1 genetic polymorphisms in prostate cancer patients further supports the notion that defective DNA repair may be associated with prostate cancer risk. Taken together these data suggest that reactive oxygen species and oxidative DNA damage may play a critical role in the development of prostate cancer. Oxidative DNA damage has been shown to be higher in the mitochondrial than in the nuclear genome due to the higher metabolic rate in the mitochondria relative to the nucleus. DNA lesions caused by ROS are numerous and include: DNA strand breaks, apurinic/apyrimidinic (AP) sites, modified DNA bases and DNA-protein cross-links. Thymine glycol (TG), 8-oxoG and other DNA base lesions may lead to deleterious biological consequences. TG is a cytotoxic lesion that blocks both DNA replication [21] and transcription [22], causing cell death. On the other hand, 8-oxoG is a pre-mutagenic lesion that results in GC to TA transversions, whereas, 8-oxoA causes both AT to GC transition and AT to CG transversion mutations [24]. Indeed, spontaneous transversion mutations have been observed in proto-oncogenes and the tumor suppressor gene, p53, a commonly mutated gene in cancer that has been shown to play a role in DNA repair. Oxidatively induced mutations in the mtDNA can lead to cellular dysfunction and have been implicated in degenerative diseases, cancer and aging. This damage must be repaired in order to maintain proper genetic integrity. Cells utilize BER pathway as the primary means of minimizing the deleterious effects associated with oxidative DNA damage. In addition to DNA repair, cells express antioxidants that detoxify ROS produced during aerobic respiration. The genome of cancer cells is more prone to oxidative damage due to the high rate of metabolism associated with increased cellular proliferation. This high metabolic rate in cancer cells may result in increased production of ROS that could significantly increase oxidative DNA damage and may be accompanied by alterations in antioxidant levels. It is plausible that increased oxidative DNA damage coupled with alteration in antioxidant levels in cancer cells may result in insufficient DNA repair. Indeed, this is the case in a few cancer cell lines studied showing defective BER in breast cancer cell lines. Recently, defects in DNA mismatch repair (MMR), the pathway that removes errors arising during DNA replication, have been reported in prostate cancer. Although, levels of oxidative DNA damage have been shown to be increased in cancerous cells or tissues relative to non-cancerous cells or tissues, the relationship between elevated oxidative DNA damage and DNA repair in carcinogenesis is still poorly understood. To our knowledge, there have been no reports addressing the repair of oxidatively induced DNA base lesions in prostate cancer cells. Therefore, we hypothesize that oxidative DNA repair may be defective in prostate tissue as a result of uncontrolled oxidative stress and increased oxidative DNA damage. We used two well-studied prostate cancer cell lines, PC-3, DU-145 and a non-malignant prostate cell line, RWPE-1 to address our hypothesis.